Background: Docetaxel resistance remains a major obstacle in the treatment of non-small cell lung cancer (NSCLC). High-mobility group box 1 (HMGB1) has been shown to promote autophagy protection in response to antitumor therapy, but the exact molecular mechanism underlying HMGB1-mediated autophagy has not been clearly defined.

Methods: Lung adenocarcinoma (LAD) cells were transfected with pcDNA3.1-HMGB1 or HMGB1 shRNA, followed by docetaxel treatment. Cell viability and proliferation were tested by MTT assay and colony formation assay, respectively. Annexin V flow cytometric analysis and western blot analysis of activated caspase3 and cleaved PARP were used to evaluate apoptosis, while immunofluorescence microscopy and transmission electron microscopy were applied to assess autophagy activity. The formation of the Beclin-1-PI3K-III complex was examined by immunoprecipitation analysis. NOD/SCID mice were inoculated with docetaxel-resistant SPC-A1/DTX cells transfected with control or HMGB1 shRNA.

Results: HMGB1 translocated from the nucleus to the cytoplasm in LAD cells exposed to docetaxel and acted as a positive regulator of autophagy, which inhibited apoptosis and increased drug resistance. Suppression of HMGB1 restored the sensitivity of LAD cells to docetaxel both in vivo and in vitro. Mechanistic investigation revealed that HMGB1 promoted the formation of the Beclin-1-PI3K-III complex through activating the mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating autophagosome formation.

Figure 7: HMGB1 promoted Beclin-1-PI3K-III core complex formation through the MEK/ERK1/2 pathway. (A) SPC-A1 cells were transfected with pcDNA3.1-HMGB1, PI3K-III siRNA or both for 48 h. Total cell lysates were subjected to western blot analysis of LC3, p62, p-ERK1/2 and GAPDH (as a loading control). (B, E) SPC-A1 cells were pretreated with PD098059 (20 μg/ml, 45 min), followed by transfection with control or pcDNA3.1-HMGB1. Whole cell lysates were analyzed by (B) western blot against LC3, p62, and p-ERK1/2 and (E) immunoprecipitation (IP) as described in Materials and methods. (C) SPC-A1 cells were transfected with pcDNA3.1-HMGB1, MEK siRNA or both for 48 h. Total cell lysates were examined by western blot analysis using specific antibodies against LC3, p62, MEK and p-ERK1/2. (D, F) SPC-A1 cells were transfected with control or HMGB1 shRNA, and then infected with the constitutively active MEK1 construct, MEK1-DD. Total cell lysates were analyzed by (D) western blot for LC3, p62 and p-ERK1/2 and (F) IP. The blots shown are representative of three separate experiments in which similar results were observed.

Mentions:
As shown in Figure 5B, we found that 3-MA, a potent PI3K inhibitor, markedly inhibited HMGB1-induced LC3-II conversion. Thus, we tested whether PI3K-III, which had been reported to help promote autophagy initiation[4,23], was required for HMGB1-induced autophagy. To exclude the effect of 3-MA on other kinases and cellular processes, siRNA specifically targeting PI3K-III was transfected into SPC-A1 cells. Figure 7A showed that reduction of PI3K-III expression in these cells distinctly inhibited HMGB1-induced autophagy, implying that PI3K-III is required for HMGB1-mediated autophagy.We next evaluated the role of the MEK-ERK1/2 pathway in HMGB1-induced autophagy, in which MEK functions as an immediate upstream activator of ERK1/2. Transfection of SPC-A1 cells with an HMGB1 cDNA plasmid activated the MEK-ERK1/2 pathway by increasing the phosphorylation of ERK1/2. However, pharmacological disruption of the MEK-ERK1/2 pathway by the specific inhibitor PD098059 that prevents ERK1/2 activation (Figure 7B) or by transfection with MEK siRNA (Figure 7C) reduced HMGB1-induced LC3-II formation and p62 degradation. Notably, suppression of PI3K-III also inhibited HMGB1-induced phosphorylation of the MEK-ERK1/2 pathway, suggesting that MEK-ERK1/2 is a downstream signal from PI3K-III (Figure 7A). Moreover, transfection of SPC-A1 cells with MEK1-DD, the constitutively active form of MEK1, an upstream activator of ERK, relieved autophagy inhibition mediated by HMGB1 (Figure 7D).

Figure 7: HMGB1 promoted Beclin-1-PI3K-III core complex formation through the MEK/ERK1/2 pathway. (A) SPC-A1 cells were transfected with pcDNA3.1-HMGB1, PI3K-III siRNA or both for 48 h. Total cell lysates were subjected to western blot analysis of LC3, p62, p-ERK1/2 and GAPDH (as a loading control). (B, E) SPC-A1 cells were pretreated with PD098059 (20 μg/ml, 45 min), followed by transfection with control or pcDNA3.1-HMGB1. Whole cell lysates were analyzed by (B) western blot against LC3, p62, and p-ERK1/2 and (E) immunoprecipitation (IP) as described in Materials and methods. (C) SPC-A1 cells were transfected with pcDNA3.1-HMGB1, MEK siRNA or both for 48 h. Total cell lysates were examined by western blot analysis using specific antibodies against LC3, p62, MEK and p-ERK1/2. (D, F) SPC-A1 cells were transfected with control or HMGB1 shRNA, and then infected with the constitutively active MEK1 construct, MEK1-DD. Total cell lysates were analyzed by (D) western blot for LC3, p62 and p-ERK1/2 and (F) IP. The blots shown are representative of three separate experiments in which similar results were observed.

Mentions:
As shown in Figure 5B, we found that 3-MA, a potent PI3K inhibitor, markedly inhibited HMGB1-induced LC3-II conversion. Thus, we tested whether PI3K-III, which had been reported to help promote autophagy initiation[4,23], was required for HMGB1-induced autophagy. To exclude the effect of 3-MA on other kinases and cellular processes, siRNA specifically targeting PI3K-III was transfected into SPC-A1 cells. Figure 7A showed that reduction of PI3K-III expression in these cells distinctly inhibited HMGB1-induced autophagy, implying that PI3K-III is required for HMGB1-mediated autophagy.We next evaluated the role of the MEK-ERK1/2 pathway in HMGB1-induced autophagy, in which MEK functions as an immediate upstream activator of ERK1/2. Transfection of SPC-A1 cells with an HMGB1 cDNA plasmid activated the MEK-ERK1/2 pathway by increasing the phosphorylation of ERK1/2. However, pharmacological disruption of the MEK-ERK1/2 pathway by the specific inhibitor PD098059 that prevents ERK1/2 activation (Figure 7B) or by transfection with MEK siRNA (Figure 7C) reduced HMGB1-induced LC3-II formation and p62 degradation. Notably, suppression of PI3K-III also inhibited HMGB1-induced phosphorylation of the MEK-ERK1/2 pathway, suggesting that MEK-ERK1/2 is a downstream signal from PI3K-III (Figure 7A). Moreover, transfection of SPC-A1 cells with MEK1-DD, the constitutively active form of MEK1, an upstream activator of ERK, relieved autophagy inhibition mediated by HMGB1 (Figure 7D).

Background: Docetaxel resistance remains a major obstacle in the treatment of non-small cell lung cancer (NSCLC). High-mobility group box 1 (HMGB1) has been shown to promote autophagy protection in response to antitumor therapy, but the exact molecular mechanism underlying HMGB1-mediated autophagy has not been clearly defined.

Methods: Lung adenocarcinoma (LAD) cells were transfected with pcDNA3.1-HMGB1 or HMGB1 shRNA, followed by docetaxel treatment. Cell viability and proliferation were tested by MTT assay and colony formation assay, respectively. Annexin V flow cytometric analysis and western blot analysis of activated caspase3 and cleaved PARP were used to evaluate apoptosis, while immunofluorescence microscopy and transmission electron microscopy were applied to assess autophagy activity. The formation of the Beclin-1-PI3K-III complex was examined by immunoprecipitation analysis. NOD/SCID mice were inoculated with docetaxel-resistant SPC-A1/DTX cells transfected with control or HMGB1 shRNA.

Results: HMGB1 translocated from the nucleus to the cytoplasm in LAD cells exposed to docetaxel and acted as a positive regulator of autophagy, which inhibited apoptosis and increased drug resistance. Suppression of HMGB1 restored the sensitivity of LAD cells to docetaxel both in vivo and in vitro. Mechanistic investigation revealed that HMGB1 promoted the formation of the Beclin-1-PI3K-III complex through activating the mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating autophagosome formation.